DNA-binding domains of Fos and Jun do not induce DNA curvature: an investigation with solution and gel methods.
We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos-Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1-4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos-Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos-Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos-Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos-Jun-DNA complexes, in which the A-tract bend is positioned close (1-2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos-Jun-DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos-Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos-Jun-DNA complexes are not indicative of (dbd)Fos-Jun-induced DNA bending (upper limit 5 degrees) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.
Sitlani
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A
Department of Chemistry, 225 Prospect Street, Yale University, New Haven, CT 06511, USA.
Crothers
D M
DM
D002474:Cell-Free System; D017364:Collagenases / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000648:ultrastructure*; D004270:DNA, Circular; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry*; D012996:Solutions; D013329:Structure-Activity Relationship; D018808:Transcription Factor AP-1 / Q000737:chemistry*
D004270:DNA, Circular; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012996:Solutions; D018808:Transcription Factor AP-1; D004247:DNA; D017364:Collagenases; D008274:Magnesium